Reaction mass of 3,7-dimethyl-1-octyl propionate and citronellyl propionate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 October 2020 - 21 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Reaction mass of 3,7-dimethyloct-6-en-1-yl propanoate and 3,7-dimethyloctyl propanoate

EC Number:

907-873-5

Molecular formula:

C13H24O2 C13H26O2

IUPAC Name:

Reaction mass of 3,7-dimethyloct-6-en-1-yl propanoate and 3,7-dimethyloctyl propanoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 20 EKIN 043, 20 EKIN 046 and 20 EKIN 051)

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Adult human donors
- Absence verified of HIV1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs, bacteria, fungus and mycoplasma.
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Expiration date: 26 October 2020 (20 EKIN 043), 16 November 2020 (20 EKIN 046) and 21 December 2020 (20 EKIN 051)

TEMPERATURE USED FOR TEST SYSTEM
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, at 37.0 ± 1.0°C (actual range 36.4 - 37.3°C)
- Humid atmosphere of 80-100% (actual range 75 - 94%) containing 5.0 ± 0.5% CO2 in air in the dark

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.

TEST FOR COLOR INTERFERENCE BY THE TEST ITEM
- To assess the color interference, 50 μL of the test item or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water.
- The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark.
- 50 μL of the test item or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL
isopropanol.
- The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking.
- If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

TEST FOR REDUCTION OF MTT BY THE TEST ITEM
- 50 μL of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline).
- The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark.
- A negative control, 50 μL sterile Milli-Q water was tested concurrently.
- If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤1.5) and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

INTERPRETATION (See Table 1)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Twenty-five μL of the undiluted test item on top of the skin tissues

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 1 hours at 37°C

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

82

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

45

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

126

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

FIRST EXPERIMENT
Skin irritation is expressed as the remaining cell viability after exposure to the test item. In the first experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 82% (117%, 117% and 12%). Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment it is considered to be nonirritant. However, since the viabilities were spread over two categories the test was repeated.

SECOND EXPERIMENT
In the second experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 45% (21%, 39% and 76%). The relative tissue viabilities for the test item were again spread over two categories. Therefore, a third experiment was performed.

THIRD EXPERIMENT
In the third experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 126% (128%, 128% and 123%). Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

ACCEPTABILITY CRITERIA
The positive control had a mean cell viability of 3.7%, 8.7% and 6.7% (experiment 1, 2 and 3 respectively) after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range in all three experiments. In the initial experiment, the standard deviation value of the percentage viability of three tissues treated with the negative or positive control was ≤ 4.4%, indicating that the test system functioned properly. The standard deviation value of the percentage viability of three tissues treated with the test item was 61% which is above the maximum of 18%, but a repeat experiment was performed, since the viability of the test item treated tissues was spread over two different categories.
In the second experiment, the standard deviation value of the percentage viability of three tissues treated with the negative or positive control was ≤ 9.1%. The standard deviation value of the percentage viability of three tissues treated with the test item was 28%, due to the results again being spread over two categories.
In the third experiment, the standard deviation value of the percentage viability of three tissues treated identically was ≤ 2.8%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 2. Mean Absorption in the In Vitro Skin Irritation Test with Citronellyl Propionate

First experiment:

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.037	1.054	1.127	1.072	±	0.048
Test item	1.249	1.257	0.128	0.878	±	0.650
Positive control	0.047	0.027	0.046	0.040	±	0.011

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.04373). Isopropanol was used to measure the background absorption.

Second experiment:

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.912	0.925	0.782	0.873	±	0.079
Test item	0.181	0.343	0.664	0.396	±	0.246
Positive control	0.063	0.083	0.082	0.076	±	0.011

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.04458). Isopropanol was used to measure the background absorption.

Third experiment:

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.122	1.086	1.122	1.110	±	0.021
Test item	1.417	1.423	1.366	1.402	±	0.031
Positive control	0.080	0.087	0.056	0.075	±	0.016

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.043317). Isopropanol was used to measure the background absorption.

 

Table 3. Mean Tissue Viability in the In Vitro Skin Irritation Test with Citronellyl Propionate

First experiment:

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	4.4
Test item	82	61
Positive control	3.7	1.1

 

Second experiment:

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	9.1
Test item	45	28
Positive control	8.7	1.2

 

Third experiment:

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	1.9
Test item	126	2.8
Positive control	6.7	1.5

 

Applicant's summary and conclusion

Interpretation of results:

other: No a skin irritant in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance does not cause skin irritation in the in vitro skin irritation test (OECD guideline 439).

Executive summary:

An in vitro skin irritation test according to OECD guideline 439 and in accordance with GLP principles was performed. The test item was applied undiluted (25 μL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item. In the first experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 82% (117%, 117% and 12%). Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment it is considered to be non-irritant. However, since the viabilities were spread over two categories the test was repeated. In this first experiment, the standard deviation value of the percentage viability of three tissues treated with the negative or positive control was ≤ 4.4%, indicating that the test system functioned properly. The standard deviation value of the percentage viability of three tissues treated with the test item was 61% which is above the maximum of 18%, which was another reason to repeat the test. In the second experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 45% (21%, 39% and 76%). The relative tissue viabilities for the test item were again spread over two categories. The standard deviation value of the percentage viability of three tissues treated with the negative or positive control was ≤ 9.1%. The standard deviation value of the percentage viability of three tissues treated with the test item was 28%, this being > 18% too. Therefore, a third experiment was performed. In the third experiment, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 126% (128%, 128% and 123%). Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant. The positive control had a mean cell viability of 3.7%, 8.7% and 6.7% (experiment 1, 2 and 3 respectively) after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range in all three experiments. In the third and final experiment, the standard deviation value of the percentage viability of three tissues treated identically was ≤ 2.8%, indicating that the test system functioned properly. In conclusion, the substance does not cause skin irritation in the in vitro skin irritation test.